Abstract 3423: RRP1B, a novel metastasis suppressor, interacts with mRNA splicing factors and regulates alternative mRNA splicing

Abstract

Abstract RRP1B (ribosomal RNA processing 1 homolog B) is a poorly characterized nuclear protein that physically interacts with the metastasis modifier SIPA1. It has previously been shown that RRP1B activation correlates with increased survival in multiple breast cancer gene expression datasets. Furthermore, human RRP1B contains a non-synonymous coding polymorphism, the variant allele of which is reproducibly associated with a lower likelihood of metastasis in over 2,000 breast cancer patients. Here, we used short hairpin RNAs to stably knockdown Rrp1b in the highly metastatic Mvt-1 cell line. Orthotopic implantation of Rrp1b knockdown cells into the mammary fat pads of syngenic FVB/NJ mice significantly increased pulmonary surface metastasis compared to controls. Stable knockdown cell lines of RRP1B were also established in MDA-MB-231 cells. Intravenous injection of these cells into the tail vein of nude mice caused an increase in pulmonary micrometastasis sites compared to controls. In addition, Rrp1b knockdown increased in vitro cell invasiveness in both Mvt-1 and MDA-MB-231 stable cell lines. Such changes in cell behavior are likely either primary events or secondary to the observed in vitro increase in cell proliferation rate following Rrp1b knockdown. RRP1B binding candidates were previously identified by tandem affinity purification and mass spectrometry. A large number of these interacting proteins were involved in alternative RNA splicing. This is a particularly interesting observation given that alternative splicing is dysregulated in advanced tumorigenesis. To verify the interaction of RRP1B and various proteins involved in RNA splicing, we performed co-immunoprecipitation and co-immunofluorescence analysis of 293ET cells transfected with either HA-tagged full length RRP1B or various RRP1B deletion mutants, as well as untransfected MDA-MB-231 cells. Through these experiments, we demonstrated that RRP1B interacts with CROP, SRFS1, PPP1A, and C1QBP, all of which are involved in alternative mRNA splicing regulation. These interactions were lost with the deletion of the RRP1B C-terminus, but not with the deletion of the highly conserved N-terminus NOP52 domain. The involvement of RRP1B in alternative splicing was confirmed with RNA-seq using the stable Mvt-1 cell lines. With the knockdown of Rrp1b, a significant change in isoform expression was observed in over 200 genes. Earlier microarray analyses demonstrated that ectopic expression of Rrp1b has profound effects on global gene expression. In addition, alternative mRNA splicing is becoming increasingly prominent in cancer progression and metastasis. Based on our current findings, we conclude that RRP1B functions as a metastasis suppressor by regulating gene expression, most likely at the mRNA splicing stage. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3423. doi:1538-7445.AM2012-3423